Myxococcus xanthus is a gram negative soil bacterium which undergoes remarkable morphogenesis forming multi-cellular fruiting bodies upon depletion of nutrient. This unique developmental feature together with its social behavior provides and excellent prokaryotic system for studying intercellular as well as intracellular communication and signal transduction during differentiation. Our recent finding that M. xanthus contains a large family of eukaryotic-like protein serine/threonine kinases also raises an interesting possibility that protein serine/threonine kinase cascades unknown in the prokaryotes play a major role in environmental adaptation and morphogenesis of M. xanthus. The wealth of knowledge on M. xanthus accumulated by our group as well as others during the past 20 years allows us to readily study this bacterium as an ideal model system for our basic understanding of differentiation and cellular communication. In this proposal, we will pursue the following specific projects: (1) Roles of protein serine/threonine kinases in M. xanthus. So far we have cloned 13 kinase genes. For some genes, the determination of their DNA sequences, identification of their gene products as kinases, and genetic and biochemical analyses of the roles of the gene products have been achieved. We will continue to work on these genes as well as others to establish primary information on their DNA sequences, genetics (deletion, mutations and lacZ fusion) and biochemistry of the enzymes. At the same time, we will initiate our effort to decipher the signalling cascades by identifying cellular substrates for individual kinases and their upstream signals. In particular, Pkn1, a developmentally produced kinase, Pkn2 and Pkn11, putative transmembrane kinases, Pkn5 and Pkn6 seemingly coregulated by sharing a 130-bp upstream fragment between the two genes, and Pkn9 expressed in both vegetative and developmental growth will be investigated. (2) Identification of protein phosphatases associated with protein kinase cascades. The existence of a large family of protein kinases suggests the requirement of phosphatases in their cascades. We will explore to identify such phosphatases by biochemical and genetic means. (3) Development-specific sigma factors. We have so far identified, SigA, the putative vegetative sigma factor, and SigB and SigC that are developmentally regulated. Recently, we have found the fourth putative sigma factor, SigD and possibly the fifth factor, SigE. In particular, we will focus our effort on SigB to elucidate its regulatory cascade-and possible interaction with kinase cascades. (4) Roles of protein W in myxospores. In the course of the study on myxospore germination, we have found the protein of 41.5 kDa to be a major protein associated with the internal structure of the myxospores. Preliminary results indicate that protein W plays an essential role in the myxospore structure. We will continue to work on this rather unusual protein for its development-specific expression, its assembly in myxospores and its functional roles. (5) Development of new genetic systems required for inducible gene expression and for establishing a strain containing multiple independent deletion mutations.